DNA ligases catalyze the final common step of DNA repair and replication by restoring the integrity of the DNA phosphodiester backbone. All known bacterial genomes encode an NAD dependent DNA ligase (LigA) which is essential for viability. Some bacterial genomes encode an additional ATP dependent ligase of unknown function. Mycobacteria, including the major human pathogen Mycobacterium tuberculosis (Mtu) and the genetically tractable M. smegmatis (MSm), encode 3 putative ATP dependent DNA ligases (LigB, LigC, LigD) in addition to LigA. The function of these ATP dependent ligases in mycobacterial DNA repair systems and mycobacterial pathogenesis is unknown. Our preliminary data shows that these 3 ligases are ATP dependent ligases in vitro, but with distinct catalytic properties. Null mutants of each ligase in MSm and Mtu indicate that these ligases are nonessential for growth individually and in combination but that three alternative ATP dependent ligases does not rescue the essentiality of LigA in M. smegmatis. In addition both ligC and ligD participate in a novel pathway of prokaryotic Non homologous end joining (NHEJ) which is efficient but low fidelity. Sequence analysis of NHEJ junctions indicates involvement of polymerase and nuclease activities in blunt end mycobacterial NHEJ. The purpose of the experiments proposed herein is to elucidate the molecular mechanisms, physiologic role, and pathogenetic importance of Non homologous end joining (NHEJ) mediated by ATP dependent DNA ligases in M. smegmatis and M. tuberculosis. We propose a multidisciplinary research program encompassing genetics, biochemistry and microbial pathogenesis that will address the hypothesis that the DNA ligases of mycobacteria are a novel DNA repair system that defends the mycobacterial chromosome against double strand breaks during in vivo growth and persistence.